Microtubules are dynamic polymers central to many processes in eukaryotic cells, including chromosome segregation, intracellular transport, and cell shape remodeling. Many of these microtubule functions are mediated by protein interactions at growing microtubule plus ends. End-binding proteins (EBs) directly recognize a structural feature of growing microtubule ends. A diverse group of proteins, called +TIPs, bind to EBs through short, conserved SxIP peptide motifs in intrinsically disordered, positively charged protein regions. These characteristics led to the identification of over thirty structurally highly heterogeneous +TIPs, but intracellular +TIP functions remain incompletely understood. A long-term goal of this project is to answer fundamental, unresolved questions of +TIP cell biology: What are molecular functions of specific +TIPs? How is microtubule plus-end-association important for these functions? How are +TIPs spatially and temporally controlled such that different +TIP complexes mediate specific microtubule activities? Based on the unexpected diversity of SxIP-motif-containing +TIPs and findings in the previous funding period, a central hypothesis of this project is that many +TIPs act as adaptors that promote spatially and temporally controlled capture of EB-positive growing microtubule ends to polarize microtubule-dependent activities, which extends the classic search-and-capture theory by providing intracellular receptors to facilitate specific interactions with EB-covered growing microtubule ends. The current application focuses on the function of CLASPs and other +TIPs that are associated with focal adhesions (FAs), multi-layered macromolecular assemblies that mediate dynamic cell interactions with the extracellular matrix (ECM). Based on preliminary data that CLASPs cluster around FAs, tether microtubules to FAs, and facilitate FA turnover, a novel mechanism is proposed in which FA-associated +TIPs establish vesicle transport tracks toward FAs to promote localized ECM remodeling and facilitate outside-in FA disassembly. Experiments in this application will define how +TIPs and microtubules control cell-matrix adhesion remodeling by employing biochemical, cell biological and advanced live cell imaging approaches: Aim 1 asks how growing and mature FAs generate a signal that recruits specific +TIPs to a zone adjacent to FAs. Aim 2 defines molecular mechanisms by which CLASPs capture and link MTs to FAs, and investigates a novel mechanism of EB regulation. Aim 3 asks how localized exocytosis promotes FA turnover, tests whether cell-matrix release is sufficient to trigger FA disassembly by developing a highly innovative light-controlled cell adhesion substrate, and analyzes the consequences of +TIP-mediated local ECM remodeling during epithelial remodeling in a physiological 3D environment. Because abnormal cell-matrix interactions contribute to cancer metastasis, and EBs are overexpressed in cancer cells indicating increased +TIP activity, in addition to establishing new paradigms relating to +TIP function, our studies are highly relevant to understanding pathological cell behavior.